The invention relates generally to methods for scaling an image, taken at a given exposure time, to a selected or different exposure time.
In the field of digital imaging of biological specimens stained with fluorescent markers, the need has arisen to compare images of the same field of view taken with different exposure times. In one instance there is a need to remove the autofluorescence from a specimen of a biological material. Specimens of biological materials typically contain substances which fluoresce over frequency ranges that overlap with those of commonly used fluorescent markers used to examine the tissue specimens for certain biological features. For instance, it is fairly common to stain specimens with antibodies to proteins of interest that are conjugated to well established fluorescent dyes such as Cy3 and Cy5. For example, human breast cancer tissue might be stained with antibodies to the p53 tumor suppression protein conjugated to Cy3.
One approach to removing autofluorescence from the microscopy image of tissue stained with such a fluorescent marker is to take an image of the same field of view before staining with the fluorescent marker and then on a pixel by pixel basis remove the signal intensity observed in the unstained image from the stained image. The desired result is a corrected image in which the fluorescent signal recorded in the channel of the fluorescent marker is just due to the binding of the fluorescent marker. However, for such a subtraction to result in an accurate image the two images should have substantially the same exposure time.
There are instances in which it is not convenient or possible to take both the autofluorescence image and the fluorescent marker stained image at the same exposure time. In general each type of image has its own optimum exposure time which gives the best balance between obtaining signal from the maximum number of pixels with avoiding the signal from any pixel saturating its recording channel and also the best balance between true signal and background signal. In some cases it may not be possible or practical to use an exposure time with some fluorescent markers as long as that desirable for measuring autofluorescence because at such long exposure times an unacceptably large number of the pixels are saturated, meaning that they are receiving so much signal that further signal is undetectable.
Therefore there is a need to estimate what the signals at the pixels of a fluorescent image would have been if the exposure time had been different. There have been attempts to do so by using the formula It2=It1*(t2/t1) wherein It1 is the intensity for exposure time t1 and It2 is the estimated intensity for exposure time t2. However, the results of such estimates have not been fully satisfactory. There have been attempts to improve upon this estimate by subtracting the background for each exposure time according to the formula It2={(It1−background at t1)*(t2/t1)}−(background at t2) but the estimates still have not been as accurate as desired.